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dc.contributor.authorRising, Kathleen A.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 57-08, Section: B, page: 5039.;Advisors: Vern L. Schramm.
dc.description.abstractThe transition state for NAD{dollar}\sp{lcub}+{rcub}{dollar} hydrolysis by the cholera toxin catalytic subunit (CTA1) has been characterized by multiple V/K kinetic isotope effects (KIE's) using labeled NAD{dollar}\sp{lcub}+{rcub}{dollar} as the substrate. CTA1 causes cholera by catalyzing the ADP-ribosylation of G{dollar}\rm\sb{lcub}s\alpha{rcub}{dollar}. In vitro, CTA1 ADP-ribosylates guanidino compounds, including free guanidine {dollar}(\rm k\sb{lcub}cat{rcub}\le 23\ min\sp{lcub}-1{rcub}, K\sb{lcub}m(NAD\sp{lcub}+{rcub}){rcub} = 14 mM){dollar}, and hydrolyzes NAD{dollar}\sp{lcub}+{rcub}{dollar} {dollar}\rm (k\sb{lcub}cat{rcub} = 8\ min\sp{lcub}-1{rcub}, K\sb{lcub}m(NAD\sp{lcub}+{rcub}){rcub} = 14 mM){dollar} to form ADP-ribose and nicotinamide. The primary {dollar}\sp{lcub}14{rcub}{dollar}C, primary {dollar}\sp{lcub}15{rcub}{dollar}N, {dollar}\alpha{dollar}-secondary {dollar}\sp3{dollar}H, {dollar}\beta{dollar}-secondary {dollar}\sp3{dollar}H, {dollar}\gamma{dollar}-secondary {dollar}\sp3{dollar}H, {dollar}\delta{dollar}-secondary {dollar}\sp3{dollar}H and primary double KIE's for NAD{dollar}\sp{lcub}+{rcub}{dollar} hydrolysis by CTA1 are {dollar}\rm 1.030\pm 0.005,\ 1.029\pm 0.004,\ 1.186\pm 0.004,\ 1.108\pm 0.004,\ 0.986\pm 0.003,\ 1.020\pm 0.003\ and\ 1.052\pm 0.004{dollar}, respectively. Compared to a family of KIE's for NAD{dollar}\sp{lcub}+{rcub}{dollar} solvolysis, these KIE's are intrinsic and provide direct information on transition state structure. These data, along with the solvent deuterium KIE of {dollar}0.82\pm 0.09{dollar} and the inability of CTA1 to catalyze NAD{dollar}\sp{lcub}+{rcub}{dollar} methanolysis, indicate that NAD{dollar}\sp{lcub}+{rcub}{dollar} hydrolysis by CTA1 proceeds through a single transition state characterized by rate-limiting cleavage of the N-glycosidic bond in a relatively desolvated environment and with minimal participation of the incoming nucleophile. The KIE's have been used to model the transition state geometry using bond-energy bond-order vibrational analysis. The transition state has substantial oxocarbonium ion character and hyperconjugation within the ribose ring and is consistent with a highly dissociative, concerted mechanism, with distances of approximately 22 A and 3.3 A from the anomeric carbon to nicotinamide and the water nucleophile, respectively. The {dollar}\gamma{dollar}- and {dollar}\delta{dollar}-secondary KIE's are unique to enzymatic stabilization of the transition state and are evidence for enzyme-substrate interactions remote from the reaction center. The {dollar}\alpha{dollar}-secondary {dollar}\sp3{dollar}H KIE for ADP-ribosyltransfer to guanidine, {dollar}1.173\pm 0.004{dollar}, indicates that the transition state for this reaction is highly dissociative, similar to that for NAD{dollar}\sp{lcub}+{rcub}{dollar} hydrolysis by CTA1.
dc.publisherProQuest Dissertations & Theses
dc.titleTransition-state analysis of cholera toxin

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